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Abstract

Introduction

The aim of this study was to examine seroconversion and the relationship with age
and inflammation of autoantibodies in a large group of patients attending an outpatient
rheumatology clinic.

Methods

Levels of antibodies to citrullinated proteins/peptides (ACPAs) and IgM rheumatoid
factor (IgM-RF) were determined in 22,427 samples collected from 18,658 patients.
The diagnosis was derived from a diagnosis registration system. The degree of seroconversion
in repeated samples and the correlation of levels with age and inflammatory markers
were determined for ACPA and IgM-RF in rheumatoid arthritis (RA) and non-RA patients.

Results

Seventy-one percent of RA patients (n = 1,524) were ACPA-positive and 53% were IgM-RF-positive;
in non-RA patients (n = 2,245), the corresponding values were 2% and 4%, respectively.
In patients with at least two samples (n = 3,769), ACPA status was more stable than
IgM-RF status in RA patients. ACPA- or IgM-RF-negative non-RA patients seldom became
positive. ACPA positivity was unrelated to age in both RA and non-RA patients. IgM-RF
positivity was unrelated to age in RA patients; however, it increased with age in
non-RA patients. The correlation between autoantibody levels and inflammatory markers
was low in general and was somewhat higher for IgM-RF than for ACPA.

Conclusions

ACPA status is more stable in time and with increasing age than IgM-RF status, further
establishing its role as a disease-specific marker. ACPA and IgM-RF levels are only
moderately correlated with markers of inflammation.

Introduction

One of the frequent characteristics of rheumatoid arthritis (RA) is the presence of
antibodies to citrullinated proteins/peptides (ACPAs) and/or IgM rheumatoid factor
(IgM-RF) [1]. IgM-RF targets the Fc fragment of IgG and is observed in about 60% to 65% of RA
patients, but it is also frequently observed in other inflammatory diseases [2,3]. ACPAs comprise a group of antibodies that are highly specific for RA: among those
are antibodies against cyclic citrullinated peptide (CCP) [4]. ACPAs target citrullinated proteins and are observed in around 70% of RA patients.
In contrast to IgM-RF, ACPA is highly specific for RA (specificity 80% versus 96%,
respectively) [3].

Besides their well-established superior specificity for RA, several other properties
of ACPA are distinct from IgM-RF. About 50% to 70% of early-RA patients are ACPA-positive,
and this phenotype remains fairly stable thereafter [2,5,6], even during treatment with tumour necrosis factor (TNF)-blocking agents [7]. On the other hand, IgM-RF levels decrease during antirheumatic treatment [8] and 17% of IgM-RF-positive RA patients turned negative after 6 months of anti-TNF
treatment [9].

Furthermore, IgM-RF [10], but not ACPA [11], is sometimes present in healthy older persons, suggesting that RF can be a consequence
of nonspecific immune activation. Moreover, it has been suggested that IgM-RF production
also is a consequence of the rheumatoid inflammation whereas ACPA may have pathophysiological
properties. Evidence supporting this concept is emerging [12]. For instance, ACPA precedes IgM-RF in the preclinical phase [13] and the change in IgM-RF levels during anti-TNF treatment is associated with the
change in acute-phase response; this is not observed for ACPA [9]. These data suggest that ACPA and IgM-RF represent two different autoantibody systems.
ACPAs are disease-specific, their presence is fairly stable in time and does not increase
with age, and ACPA levels are not correlated with the acute-phase response. On the
other hand, IgM-RF is less disease-specific, its presence increases with age in healthy/non-RA
individuals, and its levels are correlated with the acute-phase response.

Most of these data have emerged from studies of selected populations with small sample
sizes. In the present study, we sought to confirm the stability of ACPA in time, the
increased IgM-RF frequency with age, and the correlation of IgM-RF with the acute-phase
response using a repository of over 22,000 serum samples collected from over 18,000
patients attending a rheumatology clinic network in The Netherlands.

Materials and methods

ACPA and IgM-RF levels were determined in 22,427 samples, which were collected from
18,658 patients between August 2003 and August 2007. These patients attended one of
the outpatient rheumatology clinics of the Jan van Breemen Institute in the Amsterdam
region of The Netherlands. Each patient's final diagnosis was obtained from the International
Classification of Diseases version 10 diagnosis registration system, which reflects
the opinion of the treating rheumatologist. The diagnosis was categorized into five
groups according to the following codes: RA, polyarthritis or oligoarthritis, spondylarthropathy
(including ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis
associated with inflammatory bowel disease, and undifferentiated spondyloarthropathy),
osteoarthritis, and other (including arthralgia, fibromyalgia, and no final diagnosis).
The latter four groups were also combined and classified as 'non-RA'. The disease
duration at the time of autoantibody testing was variable and unknown. For the association
between age and autoantibody positivity, patients were grouped according to their
age at the first available sample: younger than 30, 30 to 39, 40 to 49, 50 to 59,
60 to 69, 70 to 79, and 80 years old or older. The local ethics committee approved
the study protocol and waived the need for informed patient consent.

Laboratory investigations

All measurements were routinely performed at the certified clinical laboratory of
the Jan van Breemen Institute. After the first sample, sequential samples were obtained
as a part of routine or protocollar care. In the case of routine care, samples were
obtained at the request of the rheumatologist at a nonspecific time point. In the
case of protocollar care, samples were obtained annually. ACPA levels were determined
by second-generation anti-CCP enzyme-linked immunosorbent assay (ELISA) (Axis-Shield,
Dundee, UK). Sera reaching 1,000 arbitrary units (AU) were not further diluted. The
cutoff level for ACPA positivity was set at 5 AU/mL in accordance with the instructions
of the manufacturer. IgM-RF levels were determined by an in-house ELISA. The cutoff
level for IgM-RF positivity was set at 30 international units (IU)/mL, which was determined
on the basis of receiver operating characteristic curves described previously [14]. Erythrocyte sedimentation rate (ESR) was measured according to the Westergren method
using a Starrsed analyser (Mechatronics, Zwaag, The Netherlands), and the reference
value was less than 15 mm per first hour. C-reactive protein (CRP) was measured on
a Cobas 6000 analyser (Roche, Woerden, The Netherlands) in accordance with the instructions
of the manufacturer, and the reference value was less than 10 mg/L.

Analysis

The effect of age on ACPA and IgM-RF positivity was determined using the chi-square
test. Correlations between levels of antibodies and levels of markers of inflammation
were determined with Spearman correlation. All analyses were performed using SPSS
16.0 software (SPSS Inc., Chicago, IL, USA).

Results

Serum samples were available in 18,658 patients: 3,116 patients with RA, 1,063 with
polyarthritis or oligoarthritis, 818 with spondylarthropathy, 2,736 with osteoarthritis,
and 10,925 classified as other. A second sample was available in 1,524 patients with
RA, 419 with polyarthritis or oligoarthritis, 195 with spondylarthropathy, 333 with
osteoarthritis, and 1,298 classified as other. Of all second samples, 35% were obtained
annually as part of a protocol, and the others were obtained according to physician
request. These 35% can be divided into patients receiving routine care (24%), described
elsewhere [15], and those receiving anti-TNF treatment (11%). In the first sample of the 18,658
patients, the percentages of patients with positive ACPA were 71% in the RA group
and 2% in the non-RA group. Rates of IgM-RF positivity were 53% in the RA group and
4% in the non-RA group. In the second sample (n = 3,769), the percentages of patients
with positive ACPA were 70% in the RA group and 7% in the non-RA group. Rates of IgM-RF
positivity were 49% in the RA group and 10% in the non-RA group.

Switch in antibody to citrullinated proteins/peptides or IgM rheumatoid factor status
between first and second samples

In patients with at least two samples, the stability of the autoantibody status was
assessed (n = 3,769). The median times between the first and second samples were similar
for RA patients (n = 1,524) and non-RA patients (n = 2,245): 11 months (interquartile
range [IQR] 4 to 13 months) and 9 months (IQR 3 to 16 months), respectively. In RA
patients, the percentages of patients switching from ACPA positivity to negativity
and from ACPA negativity to positivity were lower compared with percentage changes
in IgM-RF. In initially ACPA-positive RA patients, 1% of the second sample was negative,
whereas 13% of the second sample in IgM-RF-positive RA was negative (P < 0.001). In initially ACPA-negative RA patients, 4% of the second sample was ACPA-positive,
whereas 8% of the initially IgM-RF-negative RA patients became positive (P < 0.001). Furthermore, autoantibody-positive non-RA patients frequently became negative
in the second sample (9% for ACPA and 17% for IgM-RF, respectively), whereas autoantibody-negative
non-RA patients seldom became positive (Figure 1). When levels in RA patients were the focus, initially ACPA-positive patients showed
a nonsignificant median increase from 74 AU/mL (IQR 25 to 252) to 80 AU/mL (IQR 24
to 229), whereas in initially IgM-RF-positive RA patients, median IgM-RF levels decreased
(P < 0.001) from 94 IU/mL (IQR 51 to 188) to 81 IU/mL (IQR 41 to 178).

Figure 1. Percentage of rheumatoid arthritis (RA) and non-RA patients with a change in positivity
in antibodies to citrullinated proteins/peptides (ACPA) and IgM rheumatoid factor
(IgM-RF) between the first and second samples.

Autoantibody positivity in relation to age

To explore an effect of age on ACPA and IgM-RF positivity, we grouped patients according
to their age at the first sample. In RA, ACPA positivity was more frequent than IgM-RF
positivity in all age groups. In non-RA, however, the frequency of ACPA positivity
was lower than IgM-RF positivity in all age groups except in patients younger than
30 years old. ACPA positivity was similar among the different age groups in RA (Figure
2) as well as non-RA (Figure 3), whereas IgM-RF positivity increased with age in the non-RA group but not in the
RA group. The chi-square test for linear trend revealed a significant (P < 0.001) linear trend; IgM-RF positivity increased with age in non-RA patients.

Figure 2. Percentage of rheumatoid arthritis patients with positive antibodies to citrullinated
proteins/peptides (ACPA) or IgM rheumatoid factor (IgM-RF) status at the first sample.
Patients are grouped according to age.

Figure 3. Percentage of non-rheumatoid arthritis patients with positive antibodies to citrullinated
proteins/peptides (ACPA) or IgM rheumatoid factor (IgM-RF) status at the first sample.
Patients are grouped according to age.

Autoantibody levels in relation to markers of inflammation

In RA patients, a low correlation between autoantibodies and the levels of markers
of inflammation (as measured by ESR and CRP) was found. The correlation between IgM-RF
levels and the levels of markers of inflammation (ESR: r = 0.23; CRP: r = 0.21, both P < 0.01) was somewhat stronger compared with the correlation of ACPA levels and the
levels of markers of inflammation (ESR: r = 0.14; CRP: r = 0.14, both P < 0.01) (Table 1). In a second analysis, the correlation of the changes of levels in time of both
antibodies and markers of inflammation – that is, the correlation between (a) the
difference between the first and second ACPA/RF levels and (b) the difference between
the first and second ESR/CRP levels – was measured. The correlation between change
in ACPA levels and change of levels in markers of inflammation (ESR: r = 0.16; CRP: r = 0.13, both P < 0.01) was similar to the correlation at a single time point. For IgM-RF, the correlation
of change of levels in time with change of levels in markers of inflammation (ESR:
r = 0.31; CRP: r = 0.28, both P < 0.01) was slightly higher compared with the correlation at a single time point.
In non-RA patients, no correlation between autoantibody levels and the levels of markers
of inflammation was found at a single time point or between changes in ACPA or IgM-RF
and markers of inflammation.

Table 1. Correlations between autoantibody levels and markers of inflammation

Discussion

Characteristics of ACPA and IgM-RF were studied in a large group of RA and non-RA
patients. ACPA status was more stable than IgM-RF status in RA and non-RA patients.
ACPA positivity did not increase with age in any group, whereas IgM-RF positivity
was stable with age in RA but more frequent in older versus younger non-RA patients.
The correlation between autoantibody levels and markers of inflammation was low in
RA and absent in non-RA patients.

The results of this study show a low percentage of ACPA seroconversion in both directions
compared with IgM-RF. In very early RA, the seroconversion to positivity might occur
more frequently [5]. Previous studies of prolonged follow-up of early-arthritis patients seem to show
that qualitative changes in ACPA are rare [2,6], although the numbers of patients (n = 96 and 279) were relatively small. In RA,
ACPA seroconversion data are available from treatment cohorts, mostly with a follow-up
period of less than a year. Most, but not all, studies [16-18] reported a modest decrease in ACPA levels; however, downward seroconversion does
not seem to occur. Data on IgM-RF seroconversion in early RA are scarce. In very early
RA, a decrease in the percentage of patients positive for IgM-RF was reported [5], and in a study of early RA, 11% of the patients had variable IgM-RF status during
6-year follow-up [19]. In RA, IgM-RF downward seroconversion during anti-TNF treatment has been reported
in up to 50% of patients [7,9,20].

With regard to age and autoantibody status, ACPA positivity was stable in both RA
and non-RA, whereas IgM-RF positivity increased with age in non-RA, but not in RA.
A formal comparison cannot be made, but these results seem to be in line with earlier
observations made in healthy older persons. IgM-RF positivity increases with age;
in one study, up to 25% of persons more than 85 years old were IgM-RF-positive [10]. In a similar study, ACPA positivity was observed in only 1 out of 300 healthy individuals
over 75 years of age [11]. The fact that IgM-RF positivity increases with age in non-RA but not in RA supports
the notion that low-affinity RFs associated with infection and older age appear to
play an important role in the host response to many infectious organisms and are likely
to contribute to host defence. By contrast, high-affinity RFs in RA represent an 'autoimmune
humoral signature' that may be independent of age [21]. In a previous study reporting on IgM-RF and ACPA levels in RA, no association was
found with age [22].

The present data show modest, but significant, correlations between (changes in) ACPA
and IgM-RF levels and the inflammatory indices ESR and CRP. The correlation of changes
in autoantibody levels with changes in acute-phase markers was stronger for IgM-RF
than for ACPA, which is in line with results seen during anti-TNF treatment [9]. It has been suggested that ACPA is a disease-specific marker because of the stable
phenotype, which is confirmed in our results, and IgM-RF acts as a marker of inflammation
because it fluctuates with disease activity [9]. Observations of decreasing ACPA or IgM-RF levels were made mainly during treatment
with TNF blockers [7].

In this large population, regardless of treatment, ACPA had a low correlation with
ESR and CRP and IgM-RF also had a low correlation with ESR and CRP, although the latter
correlation was slightly higher both at a single time point and in the course of time.
Matsui and colleagues [23] reported similar correlations in a group of RA patients. The slightly stronger correlation
of IgM-RF with ESR and CRP supports the notion that IgM-RF acts as a marker of inflammation.

This study has some limitations. Diagnoses were derived from a diagnosis registration
system based on the rheumatologist's opinion, used for insurance purposes, and not
on standardized criteria. These diagnoses could change over time; the latest diagnosis
was used in the study. Furthermore, differences in disease duration and antirheumatic
treatment might have influenced these results but this information was unavailable.
Anti-TNF treatment may influence ACPA levels; however, it is not likely that this
was the cause of the observed change in ACPA positivity since a relatively small number
of patients were treated with these agents. Moreover, non-RA patients positive for
IgM-RF or ACPA might eventually develop RA since these autoantibodies are present
in the preclinical phase [13]. The strength of the study is the large number of observations from routine clinical
practice.

Conclusions

This study shows that, in a large group of rheumatology clinic patients, seroconversion
of ACPA in either direction is less frequent than that of IgM-RF. Furthermore, IgM-RF
positivity increases with age in non-RA patients, but not in RA patients, whereas
ACPA status is stable at different ages. Finally, IgM-RF levels, and to a lesser extent
ACPA levels, modestly correlate with markers of the acute-phase response.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JU performed analysis and interpretation of data and drafted the manuscript. WHB contributed
to the interpretation of data and the drafting of the manuscript. RJvdS collected
the data and was involved in the design of the study. BACD helped design the study
and draft the manuscript. DvS performed study design, interpretation of data, and
drafting of the manuscript. All authors read and approved the final manuscript.